Our overall objectives in this application are to develop a non-myeloablative allogeneic transplantation approach that requires minimal or no post-transplant immunosuppression. This approach uniquely combines hematopoietic stem cell (HSC) transplantation during the neonatal period, a positive selection strategy for in vivo amplification of genetically engineered drug-resistant donor HSC, and a negative selection strategy to eliminate these cells should an adverse event occur. The neonatal model was chosen for this approach because T cell ontogeny is incomplete at this stage and immune tolerance may be more readily achieved than with transplantation in adults. Our positive selection strategy involves lentivirus-mediated transduction of HSCs with P140K-MGMT (methylguanine-methyltransferase), a variant DMA alkyltransferase that confers resistance to endogenous MGMT inhibitors such as benzylguanine (BG) and to chloroethylating agents such as BCNU, allowing positive selection in vivo and donor cell enrichment at the stem cell level. Critical parameters for enrichment of P140K-MGMT-transduced donor HSCs after neonatal transplantation, and for induction of immunotolerance to transgene-encoded neoantigens, will first be established in a syngeneic transplant model (Aim 1). This neonatal transplantation/ in vivo positive selection strategy will then be applied to a novel semi-allogeneic non-myeloablative model that mimics HLA-haploidentical donor transplantation (Aim 2), allowing us to investigate whether enhanced levels of donor chimerism, coupled with transplantation at a stage when the host immune system is developmentally immature, will induce tolerance to graft allo-antigens. We will also test fetal liver-derived HSCs as surrogates for cord blood to determine whether the stage of donor cell ontogeny affects cellular immune responses involved in graft vs. host disease (GVHD), and examine whether the use of successive cycles of BCNU during the in vivo selection process might provide immunosuppression sufficient to limit or even abrogate GVHD. Finally, positive/negative selection vectors, in which P140K-MGMT is linked to the Herpes simplex virus thymidine kinase (HSV TK) suicide gene, will be used to assess whether ganciclovir treatment can limit GVHD by selective elimination of proliferating donor cells. This negative selection strategy may also abrogate autonomous clonal proliferation of transduced donor cells after forced enrichment and expansion in vivo, an important potential safety issue.